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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 31  |  Issue : 6  |  Page : 1198-1205
Urinary podocalyxin: Is it a real index of disease activity in egyptian patients lupus nephritis?


1 Department of Internal Medicine, Nephrology Unit, School of Medicine, Cairo University, Cairo, Egypt
2 Department of Chemical Pathology, School of Medicine, Cairo University, Cairo, Egypt

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Date of Web Publication29-Jan-2021
 

   Abstract 


Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by production of a number of antinuclear antibodies. Podocyte injury is an important feature and can be detected by several markers including podocalyxin. We aimed to evaluate the impact of SLE on urinary levels of podocalyxin and to determine its relationship to renal biopsy, proteinuria, and disease activity in lupus nephritis (LN) patients. Sixty individuals were recruited; 30 SLE patients with LN as well as 30 healthy volunteers and they were subjected to full history, clinical examination, kidney function, protein/creatinine ratio, urinary podocalyxin, and kidney biopsy. Patients with LN had higher level of urinary podocalyxin (3.96 ± 2.24) than the other group (0 ± 0), (P <0.001). Class IV LN was the most common class found among LN patients [18 cases (60%)]. There was a statistically significant positive correlation between SLE disease activity index score, protein/creatinine ratio, and urinary podocalyxin (P <0.001, r = 0.98) (P <0.001, r = 0.765). There was a statistically significant negative correlation between serum albumin, serum calcium, and urinary podocalyxin (P = 0.001, r = -0.589) (P = 0.025, r = -0.407). Urinary podocalyxin level significantly predicts the pathological impact of SLE on the kidney and could be used as a noninvasive marker for such effect and its progression.

How to cite this article:
Fayed A, Elnokeety MM, Elyamny M, Hammad H, Soliman DH, Ahmed RA. Urinary podocalyxin: Is it a real index of disease activity in egyptian patients lupus nephritis?. Saudi J Kidney Dis Transpl 2020;31:1198-205

How to cite this URL:
Fayed A, Elnokeety MM, Elyamny M, Hammad H, Soliman DH, Ahmed RA. Urinary podocalyxin: Is it a real index of disease activity in egyptian patients lupus nephritis?. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2021 Oct 26];31:1198-205. Available from: https://www.sjkdt.org/text.asp?2020/31/6/1198/308328



   Introduction Top


Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by production of a number of antinuclear antibodies.[1] Proteinuria in patients with SLE has generally been associated with immune complex deposition in the sub-epithelial and sub-endothelial glomerular capillary wall as well as endocapillary proliferation and inflammation.[2] It is a characteristic finding in all stages of lupus nephritis (LN) but is particularly characteristic of proliferative class IV and membranous class V.[1]

Regulation of the podocyte actin cytoskeleton is of critical importance for sustained function of the glomerular filter. This is mediated by several podocyte proteins such as nephrin and podocin.[3] Podocyte damage in glomerular disease is supposed to be accompanied by podocyte detachment, and shed podocytes and their fragments (marked by podocalyxin) may be identified in the urine. Urinary excretion of podocytes and/or their fragments could reflect the severity of podocyte damage and could be a useful tool in monitoring the activity of glomerular disease.[4]

Podocalyxin is an extensively O-glycosylated and sialylated type I transmembrane protein that is implicated in a wide range of cancers and is normally expressed in kidney podocytes (at the apical surface of podocytes, which faces the urinary space), hematopoietic progenitor cells, vascular endothelia, and a subset of neurons.[3]

In the current study, we aimed to evaluate the impact of SLE on urinary levels of podocalyxin and to determine its relationship with clinical disease activity, protienuria, and LN histo-pathological grading.


   Patient and Methods Top


Kasr Al-Ainy Hospital is a major hospital and a tertiary referral center serving patients from Cairo and also patients referred from all other governorates of Egypt. Patients were recruited from the outpatient Rheumatology and Clinical Immunology Clinic, Nephrology Clinic and Internal Medicine Departments during June 2017 to December 2018. They were divided into two groups: Group 1 consisted of 30 SLE patients with LN and Group 2 consisted of apparently 30 healthy volunteers of comparable age and sex to the patients' group.

The diagnosis of SLE was based on the American College of Rheumatology revised criteria for SLE.[5] The diagnosis of nephritis onset was made by either detection of elevated serum creatinine, and/or proteinuria, and/or active urine sediments. Disease activity was evaluated by the SLE Disease Activity Index (SLED AI).[6]

Exclusion criteria included any conditions or diseases known to affect endothelial function, including: smoking, diabetes mellitus, essential hypertension (HTN), known coronary artery disease, any drugs known to affect endothelial function, including nitrates, antidyslipidemic drugs, and aspirin, was withdrawn >1 week before the examination.

After discussing the procedure with each patient separately, a written consent was obtained. All our patients were subjected to full history, through clinical examination with special interest in renal involvement signs, for example, HTN, edema, and full drug history. Laboratory investigations included: routine laboratory investigations including; complete blood count, urea, serum creatinine, serum uric acid, urine analysis, protein/creatinine ratio, liver enzymes (alanine transaminase, aspartate transaminase), acute-phase reactant [erythrocyte sedimentation rate (ESR), C-reactive protein], lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, serum triglycerides). Immunological profile: anti-nuclear antibodies, anti-double stranded DNA antibodies, C3 and C4.

All renal biopsy specimens were evaluated by both light and immune fluorescence microscopes by specialized nephro-pathology team in the pathology department according to the 2003 International Society of Nephrology/ Renal Pathology Society classification.[7]

Urine (10 mL) was collected in plastic tubes, without preservative. Samples were clarified by centrifugation at 3.000 rpm for 5 min and supernatant collected in Eppendorf tubes and kept frozen at 80°C till assayed for:

  • Urine protein concentrations were measured by the Bradford method[8]
  • Estimation of urinary markers of podocyte injury: urinary podocalyxin was estimated using commercially available ELISA kits (Exocell Inc., Philadelphia, PA). Urine samples were diluted with dilution buffer provided by the ELISA kits in a ratio of 1:2 for urinary podocalyxin. Each sample was measured in duplicate. The values are expressed as ng/mL.[2]



   Statistical Analysis Top


Data were coded and entered using the IBM SPSS Statistics for Windows version 25.0 (IBM Corp., Armonk, NY, USA). Data was summarized using mean, standard deviation, median, minimum and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were made using the non-parametric Kruskal–Wallis and Mann–Whitney tests.[9] Correlations between quantitative variables were done using Spearman correlation coefficient.[10]


   Results Top


Overall, 60 females (30 patients with LN, 30 controls) with a mean age of 26.47 ± 4.49 years (18–35 years) and 25.5 ± 3.48 years (18–34 years), respectively, were included in our study. Mean duration of SLE in cases with LN was 10.21 ± 6.73 months (1–32 months). The demography and laboratory characteristics of our patients are demonstrated in [Table 1].
Table 1: The demography and laboratory characteristics of our patients.

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Renal biopsy of our patients revealed the following: Class IV LN (diffuse proliferative GN) was the most common class found among LN patients [18 cases (60%)] followed by class III (focal proliferative glomerulonephritis) and class V (membranous glomerulonephritis) with equal ratios [6 cases (20%)].

Mean of the disease activity based on SLEDAI in cases with LN were 25.33 ± 7.75 (10-40).

There were a significant lower levels of hemoglobin, lower levels of platelets, higher levels of ESR, lower levels of albumin, higher levels of urea and creatinine, and lower levels of C3 and C4 in cases of LN compared with controls with P <0.001 [Table 2].
Table 2: Comparison between cases of lupus nephritis and controls.

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There were a significant higher levels of protein/creatinine ratio and higher levels of urinary podocalyxin in cases of LN compared with controls with P <0.001 [Table 2].

There was a statistically significant positive correlation between SLEDAI score and urinary podocalyxin (P <0.001, r = 0.98). Furthermore, there was a statistically significant positive correlation between protein/ creatinine ratio and urinary podocalyxin (P <0.001, r = 0.765) [Table 3] and [Figure 1], [Figure 2].
Table 3: Correlation between urinary podocalyxin protein and different parameters.

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Figure 1: Correlation between podocalyxin in urine and Systemic Lupus Erythematosus Disease Activity Index score system for lupus activity.

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Figure 2: Correlation between podocalyxin in urine and protein/creatinine ratio.

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There was a statistically significant negative correlation between serum albumin and urinary podocalyxin (P = 0.001, r = -0.589). There was a statistically significant positive correlation between serum C4 and urinary podocalyxin (P = 0.025, r = -0.407) [Table 3].

There was a statistically significant correlation between both urinary podocalyxin levels and renal pathological class (P = 0.031) [Table 4].
Table 4: Correlation between urinary podocalyxin protein and different classes of renal biopsy.

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   Discussion Top


Proteinuria in patients with SLE has generally been associated with immune complex deposition in the sub-epithelial and sub-endothelial glomerular capillary wall as well as endo-capillary proliferation and inflammation. It is a characteristic finding in all stages of LN but is particularly characteristic of proliferative class IV and membranous class V.[1] Many studies have demonstrated the role of podocalyxin protein in the pathogenesis and progression of glomerular and tubule-interstitial injury in LN. In the current study, urinary podocalyxin was significantly higher in Group 1 (3.96 ± 2.24 ng/mL) compared to Group 2 (0 ± 0 ng/mL), (P <0.001). This result agrees with the results of previous studies which revealed that podocytes were not found in the urine of normal controls. However, all patients with clinically active LN had podocytes in their urine samples.[11]

In the current study, there was a statistically significant relation between podocalyxin in urine and the severity of lupus activity assessed by SLEDAI scoring system (P <0.001). There was a statistically significant correlation between both urinary podocalyxin levels and renal pathological class (P = 0.031). This is consistent with a study done by Nakamura et al who reported that podocytes were not found in the urine of patients without signs of systemic or renal disease. However, all patients with clinically active LN did have podocytes in their urine samples.[12]

Perysinaki et al reported that glomerular expression of nephrin and podocin significantly correlated with and became pronounced at advanced renal biopsy histopathological classes (focal and diffuse proliferative LN). The expression of nephrin and podocin was reduced in early stages of LN (mesangial).[13] This was also supported by Rezende et al.[14] Although in our study podocalyxin was found in the urine of LN patients of classes III, IV and V, there were no any class I or II patients (early stages) to compare with. In the current study, there was a statistically significant correlation between both urinary podocalyxin levels and renal pathological class (P = 0.031) This is consistent with the results of Sabino et al,[15] Wang et al[16] and Sir Elkhatim et al.[11] However, our study showed that the number of LN patients who have podocyturia in class III was six patients that was equal to that of class V and that disagree with the study done by Rezende et al who reported that a structural podocyte damage seems to occur in prolife-rative forms of LN, whereas in the pure membranous forms the predominant preserved pattern suggests a dysfunctional podocyte lesion that may account for the better long-term prognosis of proteinuria outcome.[14]

In the current study, there was a statistically significant positive correlation between albumin creatinine ratio and urinary podocalyxin protein (P <0.001, r = 0.765), this is similar to the results of the study done by Bollain-Y-Goytia et al who reported that in LN patients, reduction of glomerular podocytes significantly correlated with the cumulative excretion of urinary podocytes and proteinuria.[17]

The current study relied on the detection of podocyte related marker using ELISA estimation of its urinary level. Review of the literature detected that previous studies used ELISA estimation of urinary podocalyxin and nephrin in other kidney diseases, wherein Hara et al quantified urinary podocalyxin by ELISA in patients with glomerular diseases and patients with type 2 diabetes and found that levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes.[18]

Recently, Wang et al using indirect immuno-fluorescence, ELISA and Western blotting of urinary sediment found that urinary podocyte number and nephrin level were significantly higher in patients with LN compared to patients with other glomerular diseases and were higher in patients with severe proteinuria and urinary nephrin expression was related to podocyte and urinary albumin/creatinine ratio. Moreover, they found that urinary podocyte number and nephrin level dramatically increased in the focal segmental glomerulo-sclerosis group as compared to those of the mesangial proliferative glomerulonephritis and minimal change disease groups and concluded that the detection of the urinary podocytes and nephrin could be taken as noninvasive markers for glomerular disease severity.[19]


   Study Limitations Top


The number of individuals enrolled in our study could be a limiting factor and hence, further studies regarding this topic should be done on a larger number.


   Conclusion Top


SLE affects the glomerular structure and that can be detected by levels of urinary podocyte related protein, podocalyxin. Urinary podo-calyxin level significantly predicts the pathological impact of SLE on the kidney and could be used as a non-invasive marker for such effect and its progression.


   Ethical Committee Approval Top


The local ethical committee of the Internal Medicine department, School of Medicine, Cairo University, approved this work.


   Human and Animal Rights Top


All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.


   Informed Consent Top


Informed consent was obtained from all individual participants included in the study.

Conflict of interest: None declared



 
   References Top

1.
Gao JJ, Cai GY, Liu SW, et al. Characteristics and influence factors of pathologic transformation in the subclasses of class IV lupus nephritis. Rheumatol Int 2012;32:1751-9.  Back to cited text no. 1
    
2.
Mundel P, Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 2002;13:3005-15.  Back to cited text no. 2
    
3.
Sun D, Zhao X, Meng L. Relationship between urinary podocytes and kidney diseases. Ren Fail 2012;34:403-7.  Back to cited text no. 3
    
4.
Sekulic M, Pichler Sekulic S. A compendium of urinary biomarkers indicative of glomerular podocytopathy. Patholog Res Int 2013;2013: 782395.  Back to cited text no. 4
    
5.
Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725.  Back to cited text no. 5
    
6.
Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992;35:630-40.  Back to cited text no. 6
    
7.
Weening JJ, D’Agati VD, Schwartz MM, et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004;65:521-30.  Back to cited text no. 7
    
8.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.  Back to cited text no. 8
    
9.
Chan YH. Biostatistics 102: Quantitative data – Parametric & non-parametric tests. Singapore Med J 2003;44:391-6.  Back to cited text no. 9
    
10.
Chan YH. Biostatistics 104: Correlational analysis. Singapore Med J 2003;44:614-9.  Back to cited text no. 10
    
11.
Sir Elkhatim R, Li JY, Yong TY, Gleadle JM. Dipping your feet in the water: Podocytes in urine. Expert Rev Mol Diagn 2014;14:423-37.  Back to cited text no. 11
    
12.
Nakamura T, Ushiyama C, Suzuki S, et al. Urinary podocytes for the assessment of disease activity in lupus nephritis. Am J Med Sci 2000;320:112-6.  Back to cited text no. 12
    
13.
Perysinaki GS, Moysiadis DK, Bertsias G, et al. Podocyte main slit diaphragm proteins, nephrin and podocin, are affected at early stages of lupus nephritis and correlate with disease histology. Lupus 2011;20:781-91.  Back to cited text no. 13
    
14.
Rezende GM, Viana VS, Malheiros DM, et al. Podocyte injury in pure membranous and proliferative lupus nephritis: Distinct underlying mechanisms of proteinuria? Lupus 2014; 23:255-62.  Back to cited text no. 14
    
15.
Sabino AR, Teixeira Vde P, Nishida SK, Sass N, Mansur JB, Kirsztajn GM. Detection of podocyturia in patients with lupus nephritis. J Bras Nefrol 2013;35:252-8.  Back to cited text no. 15
    
16.
Wang Y, Yu F, Song D, Wang SX, Zhao MH. Podocyte involvement in lupus nephritis based on the 2003 ISN/RPS system: A large cohort study from a single centre. Rheumatology (Oxford) 2014;53:1235-44.  Back to cited text no. 16
    
17.
Bollain-Y-Goytia JJ, González-Castañeda M, Torres-Del-Muro F, et al. Increased excretion of urinary podocytes in lupus nephritis. Indian J Nephrol 2011;21:166-71.  Back to cited text no. 17
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18.
Hara M, Yamagata K, Tomino Y, et al. Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: Establishment of a highly sensitive ELISA to detect urinary podocalyxin. Diabetologia 2012; 55:2913-9.  Back to cited text no. 18
    
19.
Wang P, Li M, Liu Q, Chen B, Ji Z. Detection of urinary podocytes and nephrin as markers for children with glomerular diseases. Exp Biol Med (Maywood) 2015;240:169-74.  Back to cited text no. 19
    

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Correspondence Address:
Ahmed Fayed
Department of Internal Medicine, Nephrology Unit, School of Medicine, Cairo University, Cairo
Egypt
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DOI: 10.4103/1319-2442.308328

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    Abstract
   Introduction
   Patient and Methods
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